early immunocastration of pigs: from farming to meat quality
TRANSCRIPT
animals
Article
Early Immunocastration of Pigs: From Farming to Meat Quality
Daniela Werner 1 , Lisa Baldinger 1, Ralf Bussemas 1, Sinje Büttner 1, Friedrich Weißmann 1, Marco Ciulu 2 ,Johanna Mörlein 2 and Daniel Mörlein 2,*
�����������������
Citation: Werner, D.; Baldinger, L.;
Bussemas, R.; Büttner, S.; Weißmann,
F.; Ciulu, M.; Mörlein, J.; Mörlein, D.
Early Immunocastration of Pigs:
From Farming to Meat Quality.
Animals 2021, 11, 298. https://
doi.org/10.3390/ani11020298
Received: 15 December 2020
Accepted: 14 January 2021
Published: 25 January 2021
Publisher’s Note: MDPI stays neutral
with regard to jurisdictional claims in
published maps and institutional affil-
iations.
Copyright: © 2021 by the authors.
Licensee MDPI, Basel, Switzerland.
This article is an open access article
distributed under the terms and
conditions of the Creative Commons
Attribution (CC BY) license (https://
creativecommons.org/licenses/by/
4.0/).
1 Institute of Organic Farming, Johann Heinrich von Thuenen Institute, Trenthorst 32,23847 Westerau, Germany; [email protected] (D.W.); [email protected] (L.B.);[email protected] (R.B.); [email protected] (S.B.); [email protected] (F.W.)
2 Department of Animal Sciences, University of Goettingen, Kellnerweg 6, 37077 Göttingen, Germany;[email protected] (M.C.); [email protected] (J.M.)
* Correspondence: [email protected]
Simple Summary: Immunocastration of boars to prevent boar taint in meat is usually performedduring the fattening phase of pigs, which is sometimes impractical for pig fatteners. The aim ofthis study was to test the practicability of the immunization of suckling pigs against boar taintand to assess its influence on production performance and animal welfare in the fattening phase.The fattening and slaughtering performance as well as animal behavior and welfare did not differbetween the standard and the earlier immunization. However, reliable avoidance of boar taint wasnot given for all animals when immunization was conducted very early at the piglet stage.
Abstract: The study aimed to test a very early immunization of pigs to prevent boar taint with regardto its practicability and influence on production performance, its reliability in ensuring good meatand fat quality, and animal welfare. Immunization was already conducted at piglet productionstage and could be easily integrated into routine vaccination (week 3) and weaning practices (week7). The fattening and slaughter performance of the animals was not affected by the immunizationregime and was within the usual range. In addition, there were no abnormalities in animal behaviorand the prevalence of injuries caused by aggressive interactions. All animals were classified asinfertile on the basis of the histological examination of the testicles. However, the testosterone levelsmeasured at slaughter were significantly higher in animals of the early immunization regime thanin animals subjected to the standard immunization regime. Androstenone and skatole levels asthe main components of boar taint were, on average, higher and varied to a greater extent in earlyimmunized animals. Furthermore, the comparison of the immunization scheme did not result insignificant differences for meat quality and for fatty acid composition.
Keywords: meat production; performance; boar taint; animal welfare; fatty acid composition
1. Introduction
Consumers increasingly criticize pork production systems in the context of animalwelfare. Surgical castration without anesthesia, allowed in Germany until the end of 2021,is the subject of ongoing public and academic discussions. Alternatives to the surgicalcastration of pigs are the fattening of boars and immunocastration.
Immunocastration for the purpose of controlling boar taint provides several publicas well as agribusiness advantages over physical castration. Primarily, immunocastrationimproves animal welfare since it does not involve painful procedures and reduces antag-onistic, aggressive behavior by entire male pigs [1,2]. When comparing the productiveperformance of barrows to immunocastrated pigs, feed efficiency and lean carcass yieldare higher in immunocastrated animals [3], which entails economic and environmentalbenefits through reduced feed expenditures and a reduction of nitrogen emissions [4]. Theactive ingredient of the immunocastration is a protein that delays the onset of puberty
Animals 2021, 11, 298. https://doi.org/10.3390/ani11020298 https://www.mdpi.com/journal/animals
Animals 2021, 11, 298 2 of 15
by stimulating the pig’s natural immune system to produce antibodies that inhibit testesfunction. The process is based on an antigen–antibody reaction that is achieved via twoinjections of the gonadotropin-releasing hormone (GnRH)-analogon Improvac® (Zoetis,Berlin, Germany). Immunocastration requires two doses of the vaccine at least 4 weeksapart, with the second immunization usually administered 4 to 5 weeks before slaughter,which means that immunocastration is done in the fattening phase of pigs. This impliessurplus work for pig fatteners compared to surgical castration of piglets, which is espe-cially challenging when dealing with inhomogeneous pig groups, as is often the case inorganic pig fattening systems. Immunocastration at an earlier age (the first immunizationat 10 and the second at 14 weeks of age) than the recommended immunization schemehas been found to have a greater negative impact on testes structure, development, andfunction [5,6].
We have therefore tested the immunocastration in the phase of the piglet productionon the basis of the assumption that immunization at an earlier stage might lead to a perma-nent impairment of testicular development. As the needed injections for immunocastrationcan be incorporated in the standard piglet health management schemes, such a changeof time would open a new possibility for conventional and organic pig fattening systemsin terms of reduced workload while benefiting from the higher biological productivity ofimmunocastrates compared to barrows. In organic pig production systems with longersuckling periods in particular, an early immunization scheme could be easily integrated inthe operational workflow. Furthermore, an earlier immunization could reduce animal wel-fare problems due to a reduced sexual and aggressive behavior during the fattening phase.
The aim of the present study was therefore to test the practicability of the immunocas-tration at the piglet production stage, and its consequences on production performance,meat, and fat quality as well as animal behavior and welfare. To our knowledge, there areno studies with an implementation of immunocastration as early as in this study.
2. Materials and Methods
The trial was conducted at the experimental farm of Thuenen Institute of OrganicFarming in Trenthorst, Germany, between June 2018 and November 2019. Animal hus-bandry followed the rules of European Commission Regulation 889/2008. The experimentwas announced to the Schleswig Holstein Ministry of Energy Transition, Agriculture, Envi-ronment and Rural Areas on 23 April 2018 and acknowledged on 8 May 2018 (referencenumber V 244-23356/2018).
2.1. Experimental Design and Animals
Data of 109 boars from 2 treatments was obtained during 3 consecutive trial runswith both treatments being present in every trial run (see Table A1 in Appendix A). Atotal of 55 boars were objected to an early immunization scheme (EARLY) and 54 to thestandard (CONTROL) via 2 consecutive injections with the GnRH-Analogon Improvac®
(Zoetis, Berlin, Germany). Treatment groups were kept pen-wise. Piglets originated fromthe institute’s herd of crossbred sows (German Landrace × Large White), which wereinseminated with 7 individual Piétrain boars. Complete litters were assigned to 1 of the2 treatments.
All animals of CONTROL groups received the first immunization on the starting dayof the pre-fattening phase with an average group weight of 36.7 kg per animal (12 weeks).The second immunization was given 4 to 6 weeks prior to slaughter with an average groupweight of 75.3 kg (19 weeks). The pre-fattening phase ended at an average group weightof 50 kg. The subsequent fattening phase ended at a live weight of 115 kg. Animals wereclassified according to their growth capacity (slow, standard, fast) to ensure the correctimmunization dates for the second immunization. The standard class weight range wasdefined as the average weight at the start of the fattening phase ± 1 standard deviation.All animals of EARLY groups received the first immunization at 3 weeks of age (ø 6.3 kg)
Animals 2021, 11, 298 3 of 15
in combination with immunizations against mycoplasma and coli bacteria. The secondimmunization was conducted during weaning at 7 weeks of age (ø 16.4 kg).
2.2. Housing and Feeding
Animals were housed in an experimental fattening unit consisting of 4 pens with amaximum stocking rate of 10 animals per pen. Pens had solid concrete floors with straw aslitter material. Each pen had an indoor area of 1.5 m2 animal−1 including a temperature-controlled sleeping box and an automated feeding system on both sides of the pen. Theadjacent outdoor run had an area of 1.2 m2 animal−1 and was equipped with drinkingtroughs and a roughage rack.
Feeding was split in a pre-fattening and fattening phase with 2 associated compoundfeeds (pre-fattening: 88% dry matter, 19.2% crude protein, 13.2 MJ ME kg−1, 0.80 lysine/ME;fattening: 88% dry matter, 17.1% crude protein, 12.5 MJ ME kg−1, 0.74 lysine/ME). Duringthe pre-fattening phase, feeding was semi ad libitum, and during the fattening phase, aweight-oriented feeding curve was applied.
Animals had free access to clover-grass silage (18.8% dry matter, 15.6% crude protein,and 13.4 MJ ME kg−1 per dry matter) with a daily allowance of 0.5 kg and 1.0 kg freshmatter animal−1 during the pre-fattening and fattening phase, respectively.
2.3. Fattening and Slaughtering Performance
The fattening performance was characterized by live weight development and feedefficiency. Individual live weights during the fattening phase were measured weekly tocalculate live weight gains. The amounts of allocated feed and feed refusals were measureddaily. Feed efficiency was calculated per group.
Slaughter animals were transported pen wise at 5:40 a.m. to the nearby (13 km) smallfamily abattoir. Animals were unloaded pen-wise and immediately slaughtered by theuse of the electrical stunning method. The slaughtering performance included carcasscharacteristics and physical meat quality characteristics (Table A1), which were individuallymeasured 24 h post mortem using the left half of the carcass. Dressing percentage wascalculated from warm carcass weight and final body weight. Lean meat percentage wascalculated via the Bonner Formula, using caliper measurements (SCAN-STAR K, Matthäus,Eckelsheim, Germany). Electrical conductivity was measured between the 13th/14th ribvia LF-star pistol (Matthäus, Eckelsheim, Germany).
The whole data recording concerning fattening and slaughtering performance fol-lowed the German guidelines for official performance testing [7].
2.4. Boar Taint and Fatty Acid Composition
For the determination of androstenone and skatole levels, we extracted subcutaneousshoulder fat (SF) 1 day after the slaughter. For the determination of the fatty acid composi-tions of the intramuscular fat (IMF) and subcutaneous fat, we withdrew the loin muscle(Musculus longissimus thoracis et lumborum (LTL)) at the 13th rib. Shoulder fat and LTLsamples were vacuum-packed and stored at −20 ◦C until further analysis.
2.4.1. Androstenone and Skatole
Chemical analysis of androstenone and skatole was performed using stable isotopedilution analysis with headspace solid-phase microextraction followed by gas chromatogra-phy with mass spectrometry (SIDA–HS-SPME–GC/MS) using deuterium-labeled internalstandards [8]; the protocol and equipment as described in [9] was used. Results are givenas ng g−1 melted back fat.
2.4.2. Determination of the Fatty Acid Composition in the Intramuscular Fat andSubcutaneous Fat
Sample preparation of adipose tissue was performed as described by Liu et al. [10].Fatty acids of intramuscular fat were extracted from freeze-dried samples and derivatized
Animals 2021, 11, 298 4 of 15
according to the procedure by Du et al. [11] for triglycerides adopting a derivatization timeof 1 h instead of 40 min.
Fatty acid methyl esters were analyzed by gas chromatography with flame ionizationdetector (GC-FID). The gas chromatograph (GC; TRACE 1310) was equipped with anAS1310 autosampler; equipment was sourced from Thermo Fischer Scientific (Waltham,MA, USA). A Supelcowax−10 (30 m × 0.32 mm × 0.25 µm; Sigma-Aldrich Chemie GmbH,Munich, Germany) capillary column was employed for the separation. Oven temperaturewas held at 160 ◦C for 1 min, increased until 220 ◦C (heating rate: 10 ◦C/min), maintainedat 220 ◦C for 3 min, increased again to 250 ◦C (heating rate: 10 ◦C/min), and as a laststep was held at 250 ◦C for 3 min. Each sample was injected adopting a 1:50 split ratio,at 250 ◦C, using hydrogen as the carrier gas with a flow rate of 1.2 mL/min. The wholechromatographic run was completed in 16 min. The FID operated at 260 ◦C with an air flowof 350 mL/min, hydrogen flow of 35 mL/min, and makeup gas flow of 40 mL/min. Fattyacids were identified using the Supelco 37 Component FAME Mix (Sigma-Aldrich, Munich,Germany) and relative areas were analyzed using the Chromeleon Chromatography DataSystem (Version 7.2 SR9; Thermo Fischer Scientific, Waltham, MA, USA). All the analyseswere performed in duplicate.
2.5. Testicular Development and Immunization Status
Testes were weighed immediately after the slaughter. To determine the fertility status,we cut 1 cm3 cubes of testes and epididymis no later than 3 h after slaughtering. Subse-quently the sample material was prepared with 4% formaldehyde solution for overnighttransport to the Clinic for Cloven-hoofed Animals of the Faculty of Veterinary Medicine,University of Leipzig. Histological analyses of the samples were conducted the followingday as described elsewhere [12]. Results were categorized into findings on testes (atrophi-cation, hypoplasia) and epididymis (azoospermia, teratozoospermia, oligospermia).
To determine the immunization status, we collected blood samples during the exsan-guination of the pigs. Blood serum samples were stored at −20 ◦C and analyzed at the endof the trial. Analyses of the testosterone level and the absolute antigen–antibody bindinglevel in the blood serum were conducted at the Department of Behavioral Physiology ofLivestock of the University of Hohenheim as described elsewhere [13].
2.6. Animal Behaviour and Welfare
Data on animal behavior was collected via direct observations at 2 dates for each trialgroup. Each observation lasted 3 h in the morning and 3 h in the afternoon. Two observersconducted the observation prior to the second immunization of the CONTROL groupanimals as an effect of the immunization is expected 2 weeks after the second immunization(the first observation at an overall mean age of 16 weeks and the second observation at anoverall mean age of 18 weeks). Only negative behavior (pushing, biting, and mounting) wascounted. Inter-observer reliability between the 2 observers was estimated by calculatingprevalence-adjusted bias-adjusted Kappa (PABAK) values for all types of behavior [14].The prevalence of scratches and penis injuries were documented as indicators of animalwelfare. Scratches were documented during the fattening phase every 2 weeks. The leftside of the body was divided into 8 parts, which were examined according to the WelfareQuality Assessment protocol for pigs [15]. Penis injuries were scored immediately afterthe slaughter, with score 0 = no bite marks, score 2 = small bite marks, score 3 = severebite marks.
2.7. Statistical Analysis
All statistical analyses were performed using SAS 9.4. PROC MIXED and the followingmodels were used for the analysis of fattening and slaughtering performance, meat qualityand fatty acid composition, testes weight, and immunization status:
(1) Live weight: yijk = µ + Immi + Trial runj + Litterk + εijk
(2) Daily gain: yijkl = µ + Immi + Trial runj + Litterk + Start weightl + εijkl
Animals 2021, 11, 298 5 of 15
(3) Feed efficiency: yi = µ + Immi + εi(4) Slaughtering performance: yijk = µ + Immi + Trial runj + Litterk + Slaughter weightm
+ εijkm
(5) Meat and fat quality: yijk = µ + Immi + Trial runj + Litterk + εijk
(6) Testes weight: yijk = µ + Immi + Trial runj + Litterk + Age at slaughtern + Slaughterweightm + εijknm
(7) Immunization status: yijk = µ + Immi + Trial runj + Litterk + Age at slaughtern + εijk
To analyze the effects of the different immunization schemes on testicular histologyand animal welfare indicators, we compared the frequencies of categories and scores usingPROC GLIMMIX (chi2 test, binomial distribution) and the following models:
(8) Histology characteristics: yijk = µ + Immi + Trial runj + εij
(9) Behavior: yijk = µ + Immi + Trial runj + Day sectiono + εijo
(10) Appearance of scratches: yi = µ + Immi + εi
where µ = overall mean, Immi = fixed effect of immunization scheme (i = CONTROL,EARLY), Trial runj = fixed effect of trial run (j = 1, 2, 3), Litterk = random effect of litter(k = individual litter), Start weightl = continuous linear effect of live weight (l = individuallive weight), Slaughter weightm = continuous linear effect of slaughter weight (m = indi-vidual slaughter weight), Age at slaughtern = continuous linear effect of age at slaughter(n = individual age at slaughter), Day sectiono = fixed effect of time of observation (o = latemorning, afternoon), ε = residual error.
Except for feed efficiency, the individual animal was the experimental unit. Feedefficiency was analyzed per pen. Litter number was recorded to include the effect of thesow on production performance.
Probability values < 0.05 were interpreted as indicating statistically significant differences.Due to the low sample size, penis injuries were evaluated descriptively.
3. Results3.1. Fattening and Slaughtering Performance
The fattening phase in this trial comprised a live weight range from 25.3 to 51.1 kg(CONTROL) and 23.4 to 50.7 kg (EARLY) in the pre-fattening phase and ended mean liveweights of 115.2 (CONTROL) and 114.1 (EARLY), respectively. No significant differenceswere found for live weight development between the trial groups. Feed conversion didnot differ significantly between the trial groups during the two fattening phases, but it diddiffer between trial runs in the fattening phase. The daily weight gain of the EARLY pigsin the pre-fatting phase was significantly higher by 41 g than that of the CONTROL pigs.Daily weight gain from start to slaughter did not differ significantly between trial groups(903 and 916 g for CONTROL and EARLY pigs, respectively).
No significant differences in the slaughtering performance were found between thetrial groups except for electrical conductivity 24 h postmortem, which was higher in theEARLY pigs.
For detailed results, see Table A2.
3.2. Boar Taint and Fatty Acid Composition
Significant interactions between the immunization scheme and trial run were foundfor androstenone and skatole (p = 0.032 and 0.001, respectively). Androstenone concen-trations in the EARLY pigs in the third trial run were lower compared to trial runs oneand two and did not differ from values obtained in the CONTROL pigs in all trial runs.Skatole concentrations in the EARLY pigs in the first trial run were the highest amongall trial groups. The other trial groups did not differ from each other (Table A3) withrespect to skatole concentrations. The distribution of individual androstenone and skatoleconcentrations showed a wider dispersion within the EARLY group in comparison with theCONTROL group (Figure 1). On the basis of cut off values of 250 ng of skatole or 1.000 ng
Animals 2021, 11, 298 6 of 15
of androstenone per grams of fat as boundaries for boar tainted meat, we found that 7%(n = 4) of the EARLY pigs would have been classified as potentially tainted.
Animals 2021, 11, x FOR PEER REVIEW 6 of 17
two and did not differ from values obtained in the CONTROL pigs in all trial runs. Skatole concentrations in the EARLY pigs in the first trial run were the highest among all trial groups. The other trial groups did not differ from each other (Table A3) with respect to skatole concentrations. The distribution of individual androstenone and skatole concen-trations showed a wider dispersion within the EARLY group in comparison with the CONTROL group (Figure 1). On the basis of cut off values of 250 ng of skatole or 1.000 ng of androstenone per grams of fat as boundaries for boar tainted meat, we found that 7% (n = 4) of the EARLY pigs would have been classified as potentially tainted.
Figure 1. Distribution of androstenone (left) and skatole (right) concentrations as measured in the shoulder fat (SF) according to the immunization scheme.
The percentage of monounsaturated (MUFA) and polyunsaturated (PUFA) fatty ac-ids in the SF and in the IMF differed between the trial groups (Figure 2). While the fat of the CONTROL pigs had a significantly higher content of MUFA than EARLY pigs, it was the other way round for PUFA content (p < 0.001 and p = 0.015, respectively). The content of saturated fatty acids (SFA) did not differ significantly between the immunization schemes. Generally, SF had a higher content of PUFA and a lower content MUFA than IMF.
Figure 1. Distribution of androstenone (left) and skatole (right) concentrations as measured in the shoulder fat (SF)according to the immunization scheme.
The percentage of monounsaturated (MUFA) and polyunsaturated (PUFA) fatty acidsin the SF and in the IMF differed between the trial groups (Figure 2). While the fat of theCONTROL pigs had a significantly higher content of MUFA than EARLY pigs, it was theother way round for PUFA content (p < 0.001 and p = 0.015, respectively). The content ofsaturated fatty acids (SFA) did not differ significantly between the immunization schemes.Generally, SF had a higher content of PUFA and a lower content MUFA than IMF.
Animals 2021, 11, x FOR PEER REVIEW 7 of 17
Figure 2. Fatty acid composition (sum of monounsaturated fatty acid (MUFA) and polyunsaturated fatty acid (PUFA), respectively) in subcutaneous fat (SF) and intramuscular fat (IMF) according to immunization scheme.
3.3. Testicular Development and Immunization Status Testes weight of the EARLY pigs was significantly higher and showed a higher var-
iation than that of the CONTROL pigs. The immunization scheme significantly influenced testicular development. While the testes of the EARLY group pigs were mainly underde-veloped (hypoplasia), the testes of the CONTROL pigs mainly showed signs of tissue shrinkage (atrophia) (see Table 1). Additionally, atrophication of the testes of the EARLY pigs was less advanced than in the CONTROL pigs (see Figure 3).
Table 1. Testes weight and development according to immunization scheme.
Immunization p-Values Control Early
Imm Trial Run Age at Slaughter Slaughter Weight LS Means n = 54 n = 55
Testes weight (g) 225 345 <0.001 0.384 <0.001 0.013 Standard error 23.61–34.55
Relative frequencies (%) n = 106 n = 108 Testes
Hypoplasia 9.4 41.7 <0.001 0.144 - - Atrophied 90.6 58.3 <0.001 0.144 - - Epididymis
Azoospermia 20.8 51.9 <0.001 0.120 - - Oligospermia 75.5 41.7 <0.001 0.649 - -
Teratozoospermia 78.3 46.3 <0.001 0.171 - - Immunization scheme (Imm).
Histological results showed that none of the pigs in either trial groups were repro-ductive. While the number and quality of sperm in the CONTROL pigs was lower (oligo- and, teratozoospemia), the percentage of pigs that developed no sperm at all (azoospemia) was higher in EARLY group pigs (see Table 1).
Figure 2. Fatty acid composition (sum of monounsaturated fatty acid (MUFA) and polyunsaturatedfatty acid (PUFA), respectively) in subcutaneous fat (SF) and intramuscular fat (IMF) according toimmunization scheme.
3.3. Testicular Development and Immunization Status
Testes weight of the EARLY pigs was significantly higher and showed a higher varia-tion than that of the CONTROL pigs. The immunization scheme significantly influenced
Animals 2021, 11, 298 7 of 15
testicular development. While the testes of the EARLY group pigs were mainly under-developed (hypoplasia), the testes of the CONTROL pigs mainly showed signs of tissueshrinkage (atrophia) (see Table 1). Additionally, atrophication of the testes of the EARLYpigs was less advanced than in the CONTROL pigs (see Figure 3).
Table 1. Testes weight and development according to immunization scheme.
Immunization p-ValuesControl Early
Imm Trial Run Age at Slaughter Slaughter WeightLS Means n = 54 n = 55
Testes weight (g) 225 345 <0.001 0.384 <0.001 0.013Standard error 23.61–34.55
Relative frequencies (%) n = 106 n = 108
TestesHypoplasia 9.4 41.7 <0.001 0.144 - -Atrophied 90.6 58.3 <0.001 0.144 - -Epididymis
Azoospermia 20.8 51.9 <0.001 0.120 - -Oligospermia 75.5 41.7 <0.001 0.649 - -
Teratozoospermia 78.3 46.3 <0.001 0.171 - -
Immunization scheme (Imm).
Animals 2021, 11, x FOR PEER REVIEW 8 of 17
Figure 3. Atrophication classification according to immunization scheme.
Testosterone levels and GnRH binding differed significantly between the trial groups and a significant interaction of immunization scheme and trial run was found in the case of testosterone. Age at slaughter had no significant effect on testosterone values, whereas GnRH binding decreased with increasing age at slaughter. Regardless of the lower testos-terone levels of EARLY pigs in the third trial run compared to the first and second run (4.58 vs. 17.80 and 11.60 ng/mL), the levels still were markedly higher than for CONTROL pigs (0.29, 0.50, and 0.21 ng/mL in the first, second, and third run respectively). Testos-terone levels in the serum were measured with a mean time span (min,max) between the second immunization and slaughter of 6 (4,9) and 17 (12,23) weeks for the CONTROL and EARLY pigs, respectively, and they showed higher variation in EARLY pigs (Figure 4).
Figure 4. Testosterone levels depending on the number of weeks from the second immunization to slaughter according to the immunization scheme.
GnRH binding was significantly higher in the CONTROL pigs by 16.5% and was influenced by the age at slaughter (see Table A4), whereas GnRH binding decreased with increasing age. Although GnRH binding of nearly 46% (n = 21) of the EARLY pigs was at the same level measured for the CONTROL pigs (>30%), the testosterone levels of the EARLY pigs showed highly variable values compared to the CONTROL group (see Figure
Figure 3. Atrophication classification according to immunization scheme.
Histological results showed that none of the pigs in either trial groups were reproduc-tive. While the number and quality of sperm in the CONTROL pigs was lower (oligo- and,teratozoospemia), the percentage of pigs that developed no sperm at all (azoospemia) washigher in EARLY group pigs (see Table 1).
Testosterone levels and GnRH binding differed significantly between the trial groupsand a significant interaction of immunization scheme and trial run was found in thecase of testosterone. Age at slaughter had no significant effect on testosterone values,whereas GnRH binding decreased with increasing age at slaughter. Regardless of thelower testosterone levels of EARLY pigs in the third trial run compared to the first andsecond run (4.58 vs. 17.80 and 11.60 ng/mL), the levels still were markedly higher than forCONTROL pigs (0.29, 0.50, and 0.21 ng/mL in the first, second, and third run respectively).Testosterone levels in the serum were measured with a mean time span (min,max) betweenthe second immunization and slaughter of 6 (4,9) and 17 (12,23) weeks for the CONTROLand EARLY pigs, respectively, and they showed higher variation in EARLY pigs (Figure 4).
Animals 2021, 11, 298 8 of 15
Animals 2021, 11, x FOR PEER REVIEW 8 of 17
Figure 3. Atrophication classification according to immunization scheme.
Testosterone levels and GnRH binding differed significantly between the trial groups and a significant interaction of immunization scheme and trial run was found in the case of testosterone. Age at slaughter had no significant effect on testosterone values, whereas GnRH binding decreased with increasing age at slaughter. Regardless of the lower testos-terone levels of EARLY pigs in the third trial run compared to the first and second run (4.58 vs. 17.80 and 11.60 ng/mL), the levels still were markedly higher than for CONTROL pigs (0.29, 0.50, and 0.21 ng/mL in the first, second, and third run respectively). Testos-terone levels in the serum were measured with a mean time span (min,max) between the second immunization and slaughter of 6 (4,9) and 17 (12,23) weeks for the CONTROL and EARLY pigs, respectively, and they showed higher variation in EARLY pigs (Figure 4).
Figure 4. Testosterone levels depending on the number of weeks from the second immunization to slaughter according to the immunization scheme.
GnRH binding was significantly higher in the CONTROL pigs by 16.5% and was influenced by the age at slaughter (see Table A4), whereas GnRH binding decreased with increasing age. Although GnRH binding of nearly 46% (n = 21) of the EARLY pigs was at the same level measured for the CONTROL pigs (>30%), the testosterone levels of the EARLY pigs showed highly variable values compared to the CONTROL group (see Figure
Figure 4. Testosterone levels depending on the number of weeks from the second immunization toslaughter according to the immunization scheme.
GnRH binding was significantly higher in the CONTROL pigs by 16.5% and wasinfluenced by the age at slaughter (see Table A4), whereas GnRH binding decreased withincreasing age. Although GnRH binding of nearly 46% (n = 21) of the EARLY pigs wasat the same level measured for the CONTROL pigs (>30%), the testosterone levels ofthe EARLY pigs showed highly variable values compared to the CONTROL group (seeFigure 5). GnRH binding showed great variation in the EARLY pigs and was negativelycorrelated to the testosterone values in the serum (r = −0.583; p < 0.001; n = 45).
Animals 2021, 11, x FOR PEER REVIEW 9 of 17
5). GnRH binding showed great variation in the EARLY pigs and was negatively corre-lated to the testosterone values in the serum (r = −0.583; p < 0.001; n = 45).
Figure 5. Testosterone levels and gonadotropin-releasing hormone (GnRH) binding according to the immunization scheme.
3.4. Animal Behaviour and Welfare The immunization scheme had no significant effect on the occurrence of antagonistic
behavior (p = 0.8955), although the counts for biting, pushing, and riding were slightly higher for the EARLY pigs (Figure 6).
Figure 6. Counts of antagonistic behavior according to the immunization scheme.
This corresponds to the results showing the relative frequencies of injuries, with the EARLY pigs showing a slightly but not significantly higher percentage of injuries in total (see Table 2). The penis rating scores for n = 2 CONTROL and n = 17 EARLY pigs could not be deducted as the penis could not be removed from the shaft. However, the majority of penis tips was intact (92.3% and 92.1% for the CONTROL and EARLY pigs, respec-tively).
Figure 5. Testosterone levels and gonadotropin-releasing hormone (GnRH) binding according to theimmunization scheme.
3.4. Animal Behaviour and Welfare
The immunization scheme had no significant effect on the occurrence of antagonisticbehavior (p = 0.8955), although the counts for biting, pushing, and riding were slightlyhigher for the EARLY pigs (Figure 6).
Animals 2021, 11, 298 9 of 15
Animals 2021, 11, x FOR PEER REVIEW 9 of 17
5). GnRH binding showed great variation in the EARLY pigs and was negatively corre-lated to the testosterone values in the serum (r = −0.583; p < 0.001; n = 45).
Figure 5. Testosterone levels and gonadotropin-releasing hormone (GnRH) binding according to the immunization scheme.
3.4. Animal Behaviour and Welfare The immunization scheme had no significant effect on the occurrence of antagonistic
behavior (p = 0.8955), although the counts for biting, pushing, and riding were slightly higher for the EARLY pigs (Figure 6).
Figure 6. Counts of antagonistic behavior according to the immunization scheme.
This corresponds to the results showing the relative frequencies of injuries, with the EARLY pigs showing a slightly but not significantly higher percentage of injuries in total (see Table 2). The penis rating scores for n = 2 CONTROL and n = 17 EARLY pigs could not be deducted as the penis could not be removed from the shaft. However, the majority of penis tips was intact (92.3% and 92.1% for the CONTROL and EARLY pigs, respec-tively).
Figure 6. Counts of antagonistic behavior according to the immunization scheme.
This corresponds to the results showing the relative frequencies of injuries, with theEARLY pigs showing a slightly but not significantly higher percentage of injuries in total(see Table 2). The penis rating scores for n = 2 CONTROL and n = 17 EARLY pigs could notbe deducted as the penis could not be removed from the shaft. However, the majority ofpenis tips was intact (92.3% and 92.1% for the CONTROL and EARLY pigs, respectively).
Table 2. Relative frequencies of injuries of different body parts according to the immunization scheme and the trial run.
Imm Trial Run p-Values
Relative Frequencies (%) of Injuries Control Early 1 2 3Imm Trial Runn = 266 n = 259 n = 200 n = 164 n = 161
Total 41.7 45.6 58.0 40.9 28.6 0.625 <0.001Ears 15.8 17.4 18.5 20.1 19.5 0.722 0.051
Head to shoulder 24.1 23.2 32.5 20.1 16.2 0.602 <0.001Shoulder to flank 14.7 19.3 24.0 14.6 10.6 0.238 0.004
Ham 6.0 5.8 11.0 4.3 1.2 0.685 0.002Legs 1.1 2.3 3.5 1.2 0.0 0.393 0.431
Foreskin 0.4 1.5 0.0 0.6 2.5 0.157 0.418Testes 0.7 0.0 0.0 1.2 0.0 0.960 0.998
Tail 2.6 3.9 4.5 0.6 4.4 0.439 0.150
Immunization scheme (Imm).
4. Discussion
The aim of this study was to test the practicability of an immunization of sucklingpigs against boar taint and its influence on production performance and animal welfare inthe fattening phase.
During the trial phase, the early immunization scheme was easily integrated in thestandard management procedures of the organic piglet production system of the researchstation and did not create an additional workload. The first immunization of the piglets atthree weeks of age was combined with the standard immunization scheme. The immu-nization at seven weeks of age could be administered during the separation process ofthe piglets during weaning. When using the same immunization scheme in conventionalsystems, the first immunization could be administered during weaning, while the secondimmunization would have to be administered during the rearing phase. Piglets toler-
Animals 2021, 11, 298 10 of 15
ated both immunizations well, and in the subsequent fattening, no additional treatmentswere necessary.
Daily weight gain during the complete fattening period did not differ significantlybetween the trial groups and was 903 and 916 g for the CONTROL and EARLY pigs,respectively. This is in accordance to the values found for immunocastrated organic pigsusing the standard immunization scheme [16] but lower than the values for conventionallykept pigs with an early immunization scheme (8 and 12 weeks of age) [17]. Althoughfeed intake during the pre-fattening phase did not differ significantly between the trialgroups, it was higher in the EARLY pigs. This led to a significantly higher daily gain ofthe EARLY pigs in the pre-fattening phase, which can be explained by the higher growthresponse of the pigs to the second immunization. After the first immunization, pigsphysiologically resemble entire male pigs, whereas after the second immunization, themetabolism changes and feed intake increases whereas feed conversion decreases [18].Carcass yield and lean meat did not differ between trial groups and are similar to valuesfound for immunocastrated pigs, [16,19,20]. Electrical conductivity was significantly higherfor the EARLY pigs; nevertheless, this did not indicate inferior meat quality in terms ofpale, soft, and exudative (PSE) meat as the time-dependent threshold value of 6 mS/cm24 h postmortem was not exceeded [21,22].
The percentage of underdeveloped testes was higher in early immunized pigs, buttestes weights of this group showed a greater variation compared to the CONTROLpigs. This is in accordance with Sauer et al. [12], who also found a greater variationfor testes weights in pigs immunized at an age of 11 weeks and 18 or 21 weeks. Insome of the EARLY pigs, an only slightly or moderately less advanced atrophication oftestes was associated with testes weights that were comparable to values for entire malepigs. They were markedly higher than testes weights of pigs in the early immunizationschemes of Sladek et al. [23], which received a first dose at 8 weeks and a second dose 8 or15 weeks prior to slaughter. The individual time span between the last immunization andslaughter in this study was 12 to 23 weeks for the EARLY group pigs and a resumptionof steroidogenesis, which is accompanied by a rise of testosterone values, might be anexplanation for higher testes weights [17]. Highly individual and variable time spans of10 to 24 weeks from the last immunization to a resumption of testes function have beendescribed earlier [24,25]. In these studies, the determined testosterone threshold in theserum for an assumed resumption of the testicular function was 0.5 ng/mL and thereforedistinctly lower than values found for the EARLY group pigs in this study. However, inaccordance to the findings of Einarsson et al. [6], the incidence of azoospermia was higherin EARLY immunized pigs, implicating a permanently affected spermatogenesis for 50%of the EARLY pigs.
Highly variable GnRH-binding percentages, which were negatively correlated totestosterone for the EARLY pigs, indicate an onset of testosterone production for someEARLY group pigs. This might be due to a diminishing effect of GnRH immunizationduring the time from the second immunization to slaughter. Testosterone values higherthan 20 ng/mL in the serum were found for animals that were slaughtered 17 weeks afterthe second immunization and later. This is in contrast to Zamaratskaia et al. [26], who foundGnRH antibody titers 22 weeks after the second immunization that were not accompaniedby a marked increase of testosterone in the plasma. Therefore, they suggested that itmight take more than four months for immunocastrated pigs to regain their reproductivepotential. Due to the high variability found for testosterone values and GnRH binding incombination with morphological and histological testes characteristics a resumption oftestes function before slaughtering for some of the EARLY pigs can be assumed.
A significant interaction between trial run and immunization scheme on testosterone,androstenone, and skatole values was found in this study, with levels being highest forthe EARLY pigs in trial run one and lowest in trial run three. The formation of skatoleand androstenone in entire male pigs are interdependent and controlled by the productionof anabolic hormones [27]. As already described above, it is possible that for the EARLY
Animals 2021, 11, 298 11 of 15
pigs in trial run one the onset of testosterone production stimulated the formation ofandrostenone and skatole to a higher extent than in trial run two and three as age atslaughter and time span between second immunization and slaughter were higher forthese pigs. Mean age at slaughter was six days higher in pigs of trial run one and the timebetween second immunization and slaughter was one week longer than for pigs of trialrun three (18 vs. 17 weeks, respectively). Furthermore, the slaughter dates in trial run onewere distributed over a longer period than in trial runs two and three. With increasingexperience regarding the immunization of piglets against boar taint in the course of theproject, the uniformity of procedures was improved over the three consecutive trial runs.This might be an explanation for the higher variation in time spans in trial run one and theresulting variations in testosterone, androstenone, and skatole. Studies on the influenceof age on the contents of androstenone and skatole in entire male pigs reveal conflictingresults. An age-related rise of androstenone was described for entire male pigs by Bonneauet al. [28]. The authors found an effect of age on androstenone levels in young, light boars,whereas in older boars the influence of body weight was more pronounced. This is incontrast to Thomsen et al. [29], who found no influence of age on androstenone levelsin boars. Zamaratskaia et al. [30] described an age-related rise in skatole values in pigsfrom 180 d upwards. However, age at slaughter had no statistically significant effect onandrostenone and skatole values in our study. There are also studies that show an influenceof season on pubertal maturation and boar taint compounds, i.e., increasing pubertalactivity and/or androstenone contents in the fat of entire male pigs with decreasing lengthof natural light exposure (see, for example, [29]). Animals of the trial runs one, two, andthree were fattened in October/November, March/April, and July/August, respectively,and androstenone values decreased from trial run one to three. However, the pigs in thisstudy would not be classified as entire male pigs on the basis of their immunization status.As described above, the time span between the first and the second immunization was thelongest in trial run one and the slaughter dates distributed over a longer period than intrial runs two and three. Therefore, an influence of slightly skewed immunization datesbetween the trial runs on androstenone values seems more likely. Hence, a combination ofa higher age at slaughter with a decreased effect of GnRH immunization during the timefrom the second immunization to slaughter on testosterone production and therefore onthe formation of androstenone and skatole is likely.
For 7% of the EARLY pigs, threshold values of 250 ng skatole and 1000 ng androstenoneper grams of melted shoulder fat were exceeded. Furthermore, the variability of measuredboar taint compound values was higher than for the CONTROL pigs. None of the CON-TROL pigs would have been classified as boar tainted. While production of sausagewith tainted meat is possible depending on processing technique and blending percent-age [31,32], the regular immunization scheme could be favorable for fresh meat productionas the early immunization scheme used in this study could not prevent the possibility ofboar tainted meat as reliable in all animals.
The contents of fatty acids found in this study are within the ranges found by otherauthors for immunocastrated pigs [16,17,33]. The percentage of MUFA and PUFA in the SFand IMF differed significantly between trial groups, while SFA percentage did not differsignificantly. This is in contrast to Zoels et al. [17], who found no differences for MUFAand PUFA in pigs immunized at 8 and 12, or 12 and 16 weeks, respectively. Nevertheless,the differences between immunization schemes are numerically small and of little practicalimportance for commercial fresh meat production. However, percentage of PUFA in SFexceeded the recommendations of a maximum content of 12 to 15% [34,35], risking cutbacksin product quality for processed products (e.g., dry fermented sausages, cured ham). Thefeeding of silage is known to have an effect on the level of PUFA in the intramuscular fat ofpigs [36,37]. The considerable PUFA levels observed can be attributable to the inclusion ofsilage in the diet.
Observer alignment for the survey of animal behavior resulted in a PABAK value of0.88, which shows high a conformity of observations according to Gunnarsson et al. [38].
Animals 2021, 11, 298 12 of 15
Problematic animal behavior did not differ significantly between trial groups but washigher for EARLY pigs. It has been unequivocally proven that immunocastration reducesaggressive and sexual behavior in pigs [2,19,39]. Andersson et al. [19] found significantlyhigher negative animal behavior in pigs subjected to the standard immunization schemeprior to the second immunization when compared to pigs early and fully immunized at 10and 14 weeks, respectively. However, these differences were eradicated after the secondimmunization of the standard immunization group. Animal behavior in our study wasobserved prior or shortly after the booster immunization of the CONTROL, and a reducingeffect of the second immunization of the CONTROL pigs on problematic behavior is likely.It is possible that this effect was already diminished in the EARLY pigs, as behavioralobservations were conducted 9 to 11 weeks after the second immunization of this group.Nevertheless, the frequency and distribution of skin lesions did not differ significantlybetween the immunization groups throughout the fattening period, indicating no increasedaggressive behavior of the EARLY pigs during the whole production period. Sexuallymotivated behavior such as riding and/or mounting did not differ between groups, andthe percentage of penile injuries did not differ between the trial groups but the proportionof penis tips that could not be removed from the shaft after slaughter was higher in theEARLY pigs (31% vs. 4%). This indicates an arrested puerperal development in the EARLYpigs as the penile frenulum, which prevents the extrusion of the penis, was not disrupted,which is usually the case during puberty [40].
5. Conclusions
An early immunization scheme could be integrated into the usual work routines ofvaccination (week 3) and weaning (week 7) of organic piglet production without negativeconsequences on production performance compared to the standard immunization scheme.Although testosterone values were higher in early immunocastrated pigs, negative animalbehavior did not increase, and on the basis of the histological examination of the testicles,we found that all animals could be classified as infertile. Even though the time spanbetween second immunization and slaughter varied between 13 and 23 weeks and led toandrostenone and skatole levels that showed both higher average values and a significantlygreater variation in early immunized animals, the percentage of boar-tainted animals inthis group was only 7%.
We therefore conclude that the implementation of an early immunization scheme atpiglet production stage is possible. Yet, the dose applied in our trial resulted in a higherfrequency boar taint than in the standard immunization scheme.
Author Contributions: Conceptualization, R.B., F.W., and D.M.; formal analysis, L.B., S.B., J.M., andD.M.; funding acquisition, L.B., R.B., F.W., and D.M.; investigation, S.B., M.C., and J.M.; methodology,R.B., S.B., and M.C.; supervision, L.B., F.W., and D.M.; visualization, D.W., J.M., and D.M.; writing—original draft, D.W. and J.M.; writing—review and editing, L.B., R.B., S.B., M.C., D.M., and F.W. Allauthors have read and agreed to the published version of the manuscript.
Funding: The authors gratefully acknowledge funding in the framework of the Project “Investigationon the exemplary implementation of a risk minimized use of entire males in organic pork productionincluding fattening, slaughtering, and processing of meat products” of the Federal Program Organicfarming and other forms of sustainable agriculture (BÖLN #2811oe143 and #2811oe144).
Institutional Review Board Statement: Animal husbandry followed the rules of European Commis-sion Regulation 889/2008. The experiment was announced to the Schleswig Holstein Ministry ofEnergy Transition, Agriculture, Environment and Rural Areas on 23 April 2018 and acknowledgedon May 8, 2018 (reference number V 244-23356/2018).
Informed Consent Statement: Not applicable.
Data Availability Statement: The data presented in this study are available on request from thecorresponding author.
Animals 2021, 11, 298 13 of 15
Acknowledgments: In memoriam apl. Ulrike Weiler (* 13 November 1956, † 26 July 2020). We aregrateful to Sybille Knöllinger, Thilo Chillon, and Ulrike Weiler (University of Hohenheim, Germany)for GnRH binding and testosterone analyses, and Tatjana Sattler and team (University of Leipzig,Germany) for the histological evaluation of testes.
Conflicts of Interest: The authors declare no conflict of interest.
Appendix A
Table A1. Number of animals per treatment and trial run.
TreatmentTrial Run
1 2 3
Control 19 16 19Early 20 15 20
39 31 39
Table A2. Fattening and slaughtering performance and fatty acid composition of intramuscular fat (IMF) and subcutaneousfat (SF) according to immunization scheme and trial run.
LS MeansImm Trial Run p-Values
Control Early 1 2 3Imm Trial Run
Live /SlaughterWeightn = 54 n = 55 n = 39 n = 31 n = 39
Feed conversion [kg feed −1 kg gain] during . . .Pre-fattening 1.99 2.00 2.09 b 1.62 a 2.29 b 0.909 <0.001 -Standard error 0.05–0.06
Fattening 2.29 2.29 2.42 b 2.22 a 2.23 a 0.901 0.015 -Standard error 0.03–0.04
Daily weight gain [g] during . . .Pre-fattening 783 824 751 a 940 b 718 a 0.047 <0.001 <0.001Standard error 14.35–19.67
Fattening 970 969 964 980 964 0.970 0.863 <0.001Standard error 16.34–24.21
Slaughtering performance
Dressing rate [%] 77.80 77.77 77.16 a 77.89 ab 78.37 b 0.918 0.041 -Standard error 0.27–0.37
Lean meat content [%] 56.51 56.69 55.32 a 56.46 a 58.02 b 0.657 <0.001 0.149Standard error 0.33–0.47
Electrical conductivity[ms cm−1] 1.74 2.00 1.77 1.85 1.91 0.035 0.721 -
Standard error 0.09–0.14Fatty acid composition
SFA IMF (%) 36.49 36.21 35.71 a 37.36 b 35.98 0.389 <0.001 -Standard error 0.23–0.32
MUFA IMF (%) 50.12 48.85 50.39 b 51.36 b 46.70 a <0.001 <0.001 -Standard error 0.34–0.46
PUFA IMF (%) 13.37 14.98 13.88 b 11.29 a 17.35 c 0.015 <0.001 -Standard error 0.46–0.61
SFA SF (%) 34.23 34.02 34.66 b 34.72 b 33.00 a 0.453 <0.001 -Standard error 0.24–0.36MUFA SF (%) 49.04 48.52 49.75 b 48.53 a 48.05 a 0.034 <0.001 -Standard error 0.17–0.24PUFA SF (%) 16.73 17.48 15.66 a 16.73 a 18.92 b 0.021 <0.001 -Standard error 0.26–0.34a,b,c superscripts lacking a common letter indicate significant differences (p < 0.05) between treatments and trial runs, respectively.
Animals 2021, 11, 298 14 of 15
Table A3. Boar taint (measured in shoulder fat) according to immunization scheme and trial run.
Interaction of Immunization(Control, Early) and Trial Run (1–3) p-Values
Control × 1 Control × 2 Control × 3 Early × 1 Early × 2 Early × 3Imm Trial Run Imm* Trial
RunLS Means n = 19 n = 16 n = 19 n = 20 n = 15 n = 20
Androstenone(ng/g fat) 72.84 a 50.00 a 50.00 a 569.5 b 363.03 b 97.65 ac <0.001 0.016 0.032
Standarderror 82.73–95.52
Skatole(ng/g fat) 14.24 a 20.56 a 32.93 a 77.52 b 41.25 a 25.79 a 0.001 0.233 0.001
Standarderror 9.49–11.03
a,b,c superscripts lacking a common letter indicate significant differences (p < 0.05) between columns.
Table A4. GnRH binding and testosterone levels according to immunization scheme and trial run (LSQ).
Imm Trial Run p-Values
Control Early 1 2 3Imm Trial Run Imm* Trial
RunAge at
SlaughterLS Means n = 45 n = 46 n = 21 n = 31 n = 39
GnRH binding(%) 46.08 29.55 35.16 39.63 38.66 <0.001 0.424 0.115 0.007
Standard error 1.76–2.66Imm* Trial run
Control × 1 Control × 2 Control × 3 Early × 1 Early × 2 Early × 3n = 10 n = 16 n = 19 n = 11 n = 15 n = 20
Testosterone(ng/mL) 0.29 a 0.50 a 0.21 a 17.80 b 11.60 b 4.58 a <0.001 0.007 0.004 0.364
Standard error 1.67–2.24
a,b different superscripts indicate significant differences (p < 0.05) between columns.
References1. Bonneau, M.; Weiler, U. Pros and Cons of Alternatives to Piglet Castration: Welfare, Boar Taint, and Other Meat Quality Traits.
Animals 2019, 9, 884. [CrossRef] [PubMed]2. Brewster, V.; Nevel, A. Immunocastration with Improvac (TM) reduces aggressive and sexual behaviours in male pigs. Appl.
Anim. Behav. Sci. 2013, 145, 32–36. [CrossRef]3. Dunshea, F.R.; Colantoni, C.; Howard, K.; McCauley, I.; Jackson, P.; Long, K.A.; Lopaticki, S.; Nugent, E.A.; Simons, J.A.; Walker,
J.; et al. Vaccination of boars with a GnRH vaccine (Improvac) eliminates boar taint and increases growth performance1. J. Anim.Sci. 2001, 79, 2524–2535. [CrossRef] [PubMed]
4. Kress, K.; Millet, S.; Labussière, É.; Weiler, U.; Stefanski, V. Sustainability of Pork Production with Immunocastration in Europe.Sustainability 2019, 11, 3335. [CrossRef]
5. Brunius, C.; Zamaratskaia, G.; Andersson, K.; Chen, G.; Norrby, M.; Madej, A.; Lundström, K. Early immunocastration of malepigs with Improvac®—Effect on boar taint, hormones and reproductive organs. Vaccine 2011, 29, 9514–9520. [CrossRef]
6. Einarsson, S.; Brunius, C.; Wallgren, M.; Lundström, K.; Andersson, K.; Zamaratskaia, G.; Rodriguez-Martinez, H. Effects of earlyvaccination with Improvac® on the development and function of reproductive organs of male pigs. Anim. Reprod. Sci. 2011, 127,50–55. [CrossRef]
7. Richtlinie für die Stationsprüfung auf Mastleistung, Schlachtkörperwert und Fleischbeschaffenheit beim Schwein. BundesverbandRind und Schwein e.V. 2019. Available online: https://www.rind-schwein.de/services/files/brs/richtlinien/20190624%20Richtlinie%20NKP%20Deckblatt%20ObenSans.pdf (accessed on 2 November 2020).
8. Fischer, J.; Elsinghorst, P.W.; Bücking, M.; Tholen, E.; Petersen, B.; Wüst, M. Development of a Candidate Reference Method for theSimultaneous Quantitation of the Boar Taint Compounds Androstenone, 3α-Androstenol, 3β-Androstenol, Skatole, and Indolein Pig Fat by Means of Stable Isotope Dilution Analysis–Headspace Solid-Phase Microextraction–Gas Chromatography/MassSpectrometry. Anal. Chem. 2011, 83, 6785–6791. [CrossRef]
9. Trautmann, J.; Meier-Dinkel, L.; Gertheiss, J.; Mörlein, D. Boar taint detection: A comparison of three sensory protocols. Meat Sci.2016, 111, 92–100. [CrossRef]
10. Liu, X.; Trautmann Mörlein, J.; Wigger, R.; Zhou, G.H.; Mörlein, D. Fatty acid composition and its association with chemical andsensory analysis of boar taint. Food Chem. 2017, 231. [CrossRef]
11. Du, M.; Ahn, D.; Sell, J. Effects of dietary conjugated linoleic acid and linoleic: Linolenic acid ratio on polyunsaturated fatty acidstatus in laying hens. Poult. Sci. 2000, 79, 1749–1756. [CrossRef]
12. Sauer, F.; Schmoll, F.; Sattler, T. Einfluss des Alters bei der zweiten Improvac®-Vakzination auf Hodengewicht, Hodenhistologieund Ebergeruchsstoffe von männlichen Mastschweinen im Vergleich zu intakten Mastebern und Kastraten. Wien. TierärztlicheMon. 2014, 101, 103–109.
Animals 2021, 11, 298 15 of 15
13. Kress, K.; Weiler, U.; Schmucker, S.; Candek-Potokar, M.; Vrecl, M.; Fazarinc, G.; Škrlep, M.; Batorek-Lukac, N.; Stefanski, V.Influence of Housing Conditions on Reliability of Immunocastration and Consequences for Growth Performance of Male Pigs.Animals 2020, 10, 27. [CrossRef] [PubMed]
14. Byrt, T.; Bishop, J.; Carlin, J.B. Bias, prevalence and kappa. J. Clin. Epidemiol. 1993, 46, 423–429. [CrossRef]15. Dalmau, A.; Velarde, A.; Scott, K.; Edwards, S.; Butterworth, A.; Veissier, I.; Keeling, L.; Overbeke, G.; Bedaux, V. Welfare Quality®
Assessment for Pigs (Sows and Piglets, Growing and Finishing Pigs); Welfare Quality®Consortium: Lelystad, The Netherlands, 2009.16. Grela, E.R.; Kowalczuk-Vasilev, E.; Klebaniuk, R. Performance, pork quality and fatty acid composition of entire males, surgically
castrated or immunocastrated males, and female pigs reared under organic system. Pol. J. Vet. Sci. 2013, 16, 7. [CrossRef]17. Zoels, S.; Reiter, S.; Ritzmann, M.; Weiß, C.; Numberger, J.; Schütz, A.; Lindner, P.; Stefanski, V.; Weiler, U. Influences of
Immunocastration on Endocrine Parameters, Growth Performance and Carcass Quality, as Well as on Boar Taint and PenileInjuries. Animals 2020, 10, 346. [CrossRef]
18. Batorek, N.; Candek-Potokar, M.; Bonneau, M.; Van Milgen, J. Meta-analysis of the effect of immunocastration on productionperformance, reproductive organs and boar taint compounds in pigs. Animal 2012, 6, 1330–1338. [CrossRef] [PubMed]
19. Andersson, K.; Brunius, C.; Zamaratskaia, G.; Lundström, K. Early vaccination with Improvac®: Effects on performance andbehaviour of male pigs. Animal 2012, 6, 87–95. [CrossRef] [PubMed]
20. Turkstra, J.; Zeng, X.; Van Diepen, J.T.M.; Jongbloed, A.; Oonk, H.; Van de Wiel, D.; Meloen, R. Performance of male pigsimmunized against GnRH is related to the time of onset of biological response. J. Anim. Sci. 2002, 80, 2953–2959. [CrossRef]
21. Weissmann, F.; Honikel, K. Zur Frage einer Referenzmethode zum Ausschluss von PSE-Fleisch in der Wareneingangskontrolle.Mitt. Bundesanst. Fur Fleischforsch. Kulmb. 1998, 1, 300–303.
22. Mörlein, D.; Link, G.; Werner, C.; Wicke, M. Suitability of three commercially produced pig breeds in Germany for a meat qualityprogram with emphasis on drip loss and eating quality. Meat Sci. 2007, 77, 504–511. [CrossRef]
23. Sladek, Z.; Prudikova, M.; Knoll, A.; Kulich, P.; Steinhauserova, I.; Borilova, G. Effect of early immunocastration on testicularhistology in pigs. Veterinární Med. 2018, 63, 18–27. [CrossRef]
24. Claus, R.; Rottner, S.; Rueckert, C. Individual return to Leydig cell function after GnRH-immunization of boars. Vaccine 2008, 26,4571–4578. [CrossRef] [PubMed]
25. Rottner, S.; Claus, R. Return of testicular function after vaccination of boars against GnRH: Consequences on testes histology.Anim. Int. J. Anim. Biosci. 2009, 3, 1279. [CrossRef]
26. Zamaratskaia, G.; Rydhmer, L.; Andersson, H.K.; Chen, G.; Lowagie, S.; Andersson, K.; Lundström, K. Long-term effect ofvaccination against gonadotropin-releasing hormone, using Improvac™, on hormonal profile and behaviour of male pigs. Anim.Reprod. Sci. 2008, 108, 37–48. [CrossRef]
27. Claus, R.; Weiler, U.; Herzog, A. Physiological aspects of androstenone and skatole formation in the boar—A review withexperimental data. Meat Sci. 1994, 38, 289–305. [CrossRef]
28. Bonneau, M.; Conseil, G.; Giovanni, F.; Mounier, A.-M.; Peignier, Y. Effects of age and live weight on fat 5α-androstenone levelsin young boars fed two planes of nutrition. Reprod. Nutr. Dév. 1987, 27, 413–422. [CrossRef] [PubMed]
29. Thomsen, R.; Edwards, S.A.; Jensen, B.B.; Rousing, T.; Sørensen, J.T. Weight and season affects androstenone and skatoleoccurrence in entire male pigs in organic pig production. Animal 2015, 9, 1577–1586. [CrossRef] [PubMed]
30. Zamaratskaia, G.; Babol, J.; Madej, A.; Squires, E.; Lundström, K. Age-related Variation of Plasma Concentrations of Skatole,Androstenone, Testosterone, Oestradiol-17β, Oestrone Sulphate, Dehydroepiandrosterone Sulphate, Triiodothyronine and IGF-1in Six Entire Male Pigs. Reprod. Domest. Anim. 2004, 39, 168–172. [CrossRef]
31. Mörlein, J.; Meier-Dinkel, L.; Gertheiss, J.; Schnäckel, W.; Mörlein, D. Sustainable use of tainted boar meat: Blending is a strategyfor processed products. Meat Sci. 2019, 152, 65–72. [CrossRef]
32. Meier-Dinkel, L.; Gertheiss, J.; Schnäckel, W.; Mörlein, D. Consumers’ perception and acceptance of boiled and fermentedsausages from strongly boar tainted meat. Meat Sci. 2016, 118, 34–42. [CrossRef]
33. Pauly, C.; Spring, P.; O’Doherty, J.V.; Ampuero Kragten, S.; Bee, G. Growth performance, carcass characteristics and meat qualityof group-penned surgically castrated, immunocastrated (Improvac®) and entire male pigs and individually penned entire malepigs. Animal 2009, 3, 1057–1066. [CrossRef] [PubMed]
34. Fischer, K.; Lindner, J.; Judas, M.; Höreth, R. Schlachtkörperzusammensetzung und Gewebebeschaffenheit von schwerenSchweinen–II. Mitteilung: Merkmale der Fleisch-und Fettqualität. Arch. Anim. Breed. 2006, 49, 279–292. [CrossRef]
35. Bee, G.; Für Nutztiere, E.F. Das Fettzahl-konforme Schwein: Die MUFA-PUFA-Norm. SchR Inst. Nutztierwiss 2004, 25, 37–51.36. Johansson, L.; Lundström, K.; Jonsäll, A. Effects of RN genotype and silage feed on fat content and fatty acid composition of fresh
and cooked pork loin. Meat Sci. 2002, 60, 17–24. [CrossRef]37. Wood, J.D.; Enser, M.; Fisher, A.V.; Nute, G.R.; Sheard, P.R.; Richardson, R.I.; Hughes, S.I.; Whittington, F.M. Fat deposition, fatty
acid composition and meat quality: A review. Meat Sci. 2008, 78, 343–358. [CrossRef] [PubMed]38. Gunnarsson, S.; Algers, B.; Svedberg, J. Description and Evaluation of a Scoring System of Clinical Health in Laying Hens.
Laying Hens in Loose Housing Systems. Ph.D. Thesis, Swedish University of Agricultural Sciences, Uppsala, Sweden, 2000. ActaUniversitatis Agriculturae Sueciae Veterinaria 73.
39. Rydhmer, L.; Lundström, K.; Andersson, K. Immunocastration reduces aggressive and sexual behaviour in male pigs. Animal2010, 4, 965–972. [CrossRef]
40. Althouse, G.C. 15 Applied Andrology in Swine. Anim. Androl. Theor. Appl. 2014, 404. [CrossRef]